Pulmonary arterial hypertension (PAH) is a progressive disease with high morbidity and mortality. Mutations and/or haploinsufficiency of BMPRII is considered to underlie familial pulmonary arterial hypertension (FPAH) as well as many instances of idiopathic PAH (IPAH). However, disease development, even in FPAH with known mutations in BMPRII, has low (10-15%) penetrance leading to searches for candidate second-hit genes that might be involved. Additionally, there is a sexual dimorphism in this disease with the incidence approximately 2.5-fold higher in women as in men. The underlying bases for low penetrance and sexual dimorphism remain unknown. At the subcellular level, endothelial and smooth muscle cells in the lung vascular lesions of PAH have an increase in cell size (megalocytosis), characteristic cytoplasmic organellar changes (Golgi apparatus fragmentation, cystic dilatation of the endoplasmic reticulum) and an inability to handle intracellular trafficking of proteins. The present project is based on our unexpected discovery that a well-known transcription factor STAT5a nongenomically associates with the Golgi membranes in the cytoplasm of pulmonary vascular cells. siRNA mediated knockdown of STAT5a/b in primary HPAECs and HPASMCs in culture produced Golgi fragmentation, cystic dilatation of endoplasmic reticulum (ER), increased Nogo-B/RTN4 and atlastin-3 (ATL3)(respectively an ER structural protein and an ER-resident GTPase), lunate distortion of nuclei, reduced membrane trafficking and secondary mitochondrial dysfunction and fragmentation - all changes that we and others (e.g. Smith & Heath, 1977, 1979) have shown to occur in PAH - changes that we show can be blocked by overexpression of the ER/Golgi-resident GTPase atlastin-1. Also, double knockdown of STAT5a and BMPRII combinatorially inhibited ER to plasma membrane trafficking in ECs suggesting second-hit effects between the two. Remarkably, it is well known that STAT5 species are estrogen-responsive and are known to mediate a sexual dimorphism phenotype. In Specific Aim I we propose to investigate the occurrence of the subcellular STAT5a/b knockdown signature in cells in vascular lesions in PAH lung tissues, identify the particular cells involved, and investigate gender-based differences in these changes. In Specific Aim II we propose to investigate endothelial and smooth muscle cells in culture derived from PAH patients for the occurrence of the subcellular STAT5a/b knockdown signature and potential gender-based differences, the combinatorial effects of STAT5a/b knockdown and BMPRII changes, and the therapeutic potential of restoring wt STAT5a and atlastin isoforms in such cells. This project represents evaluation of a new paradigm in PAH with a focus on the newly discovered cytoplasmic organellar functions of STAT5a/b as potential second-hit targets with underlying sexual dimorphism contributing to the development of this disease. PUBLIC HEALTH RELEVANCE: Pulmonary arterial hypertension (PAH) is a progressive fatal lung disease in man. We propose an all together novel way of thinking about the cause of this fatal disease. We suggest that there is a coordinated defect in the cytoplasmic organelles inside cells in disease lesions that leads to an increase in size of the cells as well as increase in number due. The defect lies in the ability of such cells to handle the movement of proteins inside the cells. The overall consequence is blockage of the blood vessels. The proposed mechanism opens up new ways to treat this disease using gene therapy. (End of Abstract)